Technical Abstract:
Salmonella enterica subsp. I serovar Enteritidis (S. Enteritidis) is the world's leading cause of salmonellosis. Eggs contaminated by apparently healthy hens and that have been improperly cooked can result in illness in humans who consume them. Although the incidence of this pathogen within the United States has not been as high as it has been in Europe, current estimates from the Centers for Disease Control and from the USDA-Food Safety Inspection Service respectively suggest that this pathogen is causing more human illness and that it has begun to contaminate broilers in the States (1). Thus, evolution impacting egg contamination is a general measure of virulence for this pathogen.
<p>Some strains of S. are monomorphic subpopulations that have the potential to complement each other in the infection pathway that results in egg contamination (2). However, other strains evolved dimorphism, which is the ability to exhibit multiple easily observed phenotypes from one genome in response to environmental conditions. To investigate the genetic basis for the emergence of subpopulation biology that alters virulence potential, the genomes of two monomorphic strains of S. Enteritidis PT13a that were descended over ten years from a single parent were subjected to virtual subtraction hybridization against dimorphic S. Enteritidis PT4 (3). Direct comparison of the closely related genomes was possible by using the genome database for S. Enteritidis PT4 NCTC 13349 to generate overlapping sets of primers (Nimblegen, Inc.) that were capable of detecting single nucleotide polymorphisms (SNPs). The database of the S. Enteritidis PT4 reference genome is from the Pathogen Sequencing Group at the Sanger Institute and can be obtained from http://www.sanger.ac.uk/Projects/Salmonella/.
<p>Part of the table lists 217 chromosomal polymorphisms detected by more than one method and part lists locations yet to be confirmed by anything other than mutational mapping (Table S1). Of these polymorphisms, 112 (51.6%) were SNPs or deletions that altered amino acid sequence or disrupted ORFs. The virulence plasmid had 3 SNPs and these differentiated the wt PT13a plasmid from the bf PT13a and reference strains without altering ORFs. The class of amino acid change introduced by SNPs was as follows: 58 neutral non-polar, 30 neutral polar, 5 basic polar and 3 acidic polar amino acids were converted in the PT4 reference genome to a different class of amino acid in one of the PT13a strains. Twenty-four (24) SNPs occurred in non-coding regions, 3 removed a premature termination codon and restored ORF read-through, and 2 introduced a premature termination codon and disrupted ORFs. There were 6 deletion events, ranging in size from 1 bp (sefD) to 215 bp (5’ end and upstream region of a gene similar to STM4551). Other genes with deletions that differentiated S. Enteritidis PT13a monomorphic strains were dsdA (10bp), STM4272 homolog (12 bp), yjfK/yjfL (11bp) and a mocR family gene (92 bp). Genes SEN0420 (STM0438), dsdA, yjfK, yjfL, sefD, ratB, and mhpC had the reference genome sequence and were linked to egg contamination. Biofilm formation of the PT4 reference strain and S. Enteritidis PT13a was linked to genes SEN1152 (STM1048), SEN2510 (STM2530), SEN3898 (mocR subfamily gene) and SEN4316 (STM4551). These results continue to support the concept that circulating strains of S. Enteritidis evolved from a hybrid parent (4).